The Possession Sound’s Snohomish River Estuary (PSSRE) provides a prime habitat for a large nesting population of North American osprey (Pandion haliaetus) in the Puget Sound region. The ospreys that nest in the Possession Sound are migratory, arriving between mid-March and mid-April to nest and leaving between late-August and late-September to winter in Southeastern Texas, Mexico, and Central America. Male and female ospreys migrate separately, with males returning first to prepare the nest and females leaving first to travel farther south. Juvenile ospreys do not make the return migration from their southern winter location until their second spring. As of 2016, osprey attendance reached 24 nesting pairs in the PSSRE. As apex predators whose diets are comprised primarily of fish, ospreys are considered sentinel species for studying contaminant levels in marine environments. Therefore, monitoring behavior, breeding success, and contaminant levels of ospreys can provide a census for the health of the local marine ecosystem. This study will take place on the return of the ospreys to the PSSRE in 2018 and will entail visually surveying the estuary to determine the nesting locations, return rates for both males and females, and dietary habits of ospreys. Past nest locations and populations will be compared to 2018 data. Additional research will utilize a remote camera with pan/tilt/zoom capabilities to observe and record three nest sites near the Port of Everett for nesting behaviors, fecundity, and identification of prey targets for potential heavy metal analysis.

Eelgrass, Zostera marina, is a critical nursery for numerous marine organisms in Puget Sound. A variety of fish species benefit from eelgrass ecosystems, using the blades of sea grass as anchor points to ensure the safety for eggs through larval fish stages. This study compared larval fish densities in eelgrass beds versus offshore from the eelgrass sites. Higher numbers of larval fish are expected to be found in samples collected within eelgrass beds rather than those collected further offshore. A variety of local fish species are expected to be found both within and outside of the eelgrass beds, including forage fish and rockfish. Samples were at three sites of eelgrass beds located in the Possession Sound in Puget Sounds’ Whidbey Basin. The samples were collected once a month from December to March with a 500-micron net deployed for three minutes at speed of 1 knot at a depth of 2 to 3 meters below the surface directly over the eelgrass bed and repeated offshore from the site. After collection, the larval fish were extracted and sent to the Shoreline Community College Laboratory for DNA barcoding to allow for species identification. Preliminary data indicates that higher numbers of larval fish were present within eelgrass beds as opposed to the offshore areas. A greater understanding of the important role that these eelgrass ecosystems play as nurseries for larval fish is necessary to provide context and further reason for conservation efforts and general awareness of human impact on these lively nearshore environments. Identification of the larval fish species that inhabit eelgrass beds provides a greater understanding of these species’ preferred laying conditions.

Epigenetic modifications regulate chromatin structure and function, playing important roles in altering DNA transcription levels and subsequently cell fate decisions. Various next-generation sequencing (NGS) methods have been developed to detect these epigenetic changes in the genome, such as chromatin immunoprecipitation followed by sequencing (ChIP-seq). Even though ChIP-seq is extensively used to analyze DNA and histone modification levels, this method is limited to one histone marker at a time and requires significant amount of input cells, which masks the profiles of cell-to-cell variation and the complex interaction between the epigenome and gene expression. To overcome these limitations in current next-generation sequencing methods, we have been developing a multiplexed assay that could detect multiple epigenetic modifications in single cells. So far, I have developed a NGS data analysis pipeline to identify potential gene candidates that are highly differentially modified by histone markers. In the future, we hope to use these gene candidates as templates to design DNA-fluorescent in situ hybridization (DNA-FISH) probes and perform Expansion Microscopy and DNA-FISH with these probes to link histone modifications to specific gene loci at high resolution. After the assay is fully developed and validated, we plan to utilize the assay to take the epigenetic profiles of hematopoietic stem cells and study cell fate decisions in hematopoiesis.

The Puget Sound is a complex estuarine system within the Salish Sea, fed by both high salinity water from the Pacific Ocean and freshwater from a number of rivers. The Snohomish River is the second largest input, transporting freshwater from the Skykomish and Snoqualmie rivers to Port Gardner Bay off the coast of Everett. At its mouth, the higher density salt water from the Puget Sound intrudes into the freshwater, forming a salt wedge that causes a highly stratified water column which rapidly changes with the tidal cycle. These mixing dynamics may be the primary driver of biological productivity in the estuarine system. To characterize the density profile relative to tidal patterns and season, current speed and velocity were recorded utilizing a Nortek ADCP. During normal outflow (3,500 f^3/s) of the Snohomish River, upward velocities were positive during flood tide and negative during ebb tide, following a typical salt-wedge trend. During a peak outflow event (56,000 f^3/s) on October 22nd, this tidal cycle dependence was disrupted and downwelling dominated in the form of negative vertical velocities throughout the water column. In addition, the magnitude of east/west velocities increased during peak outflow. This signaled a period of intense turbulence at the bottom of the water column. During the same time span, data concerning turbidity and chlorophyll levels were recorded utilizing a Seabird CTD. No clear trend was evident in chlorophyll levels at both normal and peak outflow timespans. Turbidity maintained high levels (35 NTU) following peak outflow, possibly reflecting the transportation of sediment from the river bed into the water column. This transportation would be caused by the turbulence reflected in the higher magnitude of the east/west velocity data.

Possession Sound is a salt-wedge estuary influenced by both sea water entering through the Puget Sound and fresh water entering from the Snohomish River. Regionally, upwelling off the Pacific Coast during spring and summer months brings acidic deep water into the Puget Sound. Locally in the Possession Sound, previous research has shown that seasonal cycles in both temperature and river discharge are the primary influencers of pH. These cycles are affected by the El Niño Southern Oscillation (ENSO) climate pattern, which alternates between a cooling phase (La Niña), a warming phase (El Niño), and a neutral period. The biological processes of many organisms are influenced by pH levels, and few studies have examined interannual pH variation in estuaries. This study seeks to identify the influence of ENSO on pH cycles in two different areas of Possession Sound, based on proximity to the Snohomish River. Surface pH and temperature data were analyzed at two sites, Buoy (closer to the river) and MBT (farther from the river) from January 2010 to September of 2017, using Yellow Springs Instruments and a Niskin Bottle. River discharge data was ascertained from United States Geological Survey data recorded near Monroe, Washington (2010-2017). The hypothesis of spatial variability in pH due to proximity to the river was not supported. Many of the trends at Buoy were the same or more pronounced at MBT, suggesting that the influence of the Snohomish River at Buoy did not make that site more sensitive to ENSO than MBT. Surface pH was more variable at MBT than at Buoy during the summer months, presumably due to eutrophication and subsequent decay during periods of intense biological productivity. Future studies are planned for seasonal variations of biological factors along with dissolved oxygen variation with depth to further analyze these pH trends.

Zika virus has become a major epidemic around the world due to the serious birth defects that it can cause; this has led to an increase in research regarding Zika and how to diagnose the disease. Diagnostic tests require a specific sample type to be tested for the presence of a virus, and because it has been shown that Zika viral levels are more consistent and reliable in whole blood compared to plasma or serum, whole blood is the more efficient sample type. Current strategies for diagnosing Zika fall short because there is limited data on how the virus presents itself in the body, and many of these strategies utilize expensive and time-consuming methods that are not feasible in low-resource settings where Zika is more prevalent. Therefore, there is a need for a low-cost and instrument-free process that can effectively purify Zika virus RNA from whole blood in order to allow for detection of the viral RNA. This project dealt with optimization of an existing lab protocol that required laboratory equipment and resulted in low efficiency. The aim was to develop a robust, high-efficiency, instrument-free RNA purification protocol for whole blood samples. The method of RNA purification consisted of a lysis and binding buffer, followed by RNA purification using silica beads embedded in paper to extract pure RNA from the sample. qRT-PCR was then used to quantify the resulting RNA. So far, the paper-based extraction method recovers approximately 20% of the initial target RNA; however, there are still some techniques that will be employed in the future to help improve this recovery rate. The goal of this project is to increase RNA purification efficiency and to provide an alternative mechanism to purify nucleic acids that can be used in a Zika virus diagnostic test.

Standard medical practice does not consider a lateral leaning uterus to be medically relevant. However, many indigenous cultures globally have used uterine abdominal massage to correct a “displaced” uterus to enhance fertility. A leaning uterus could theoretically interfere with ovarian function. Additionally, Research indicates that the circulatory functions, the autonomic innervations of the uterus, and pelvic vein incompetence may negatively affect ovarian function, and fertilization. This study examines a population of 30 U.S. fertility patients undergoing ovarian stimulation for an In Vitro Fertilization (IVF) procedure (an assisted reproductive medical technique used to assist with the conception of a child). We will identify differences in the quantity, and size of follicles that are produced by the ovaries of patients with a leaning uterus. At the beginning of a patient’s IVF cycle, researchers will palpate the lower abdomen to determine the position of the uterus. After the patient completes their series of gonadotropins, prescribed hormones used to stimulate follicular growth in the ovaries, physicians will record the number, size, and location of each follicle observed. We predict that a laterally leaning uterus reduces follicle number and size. If our results identify a correlation between uterine displacement and follicle development, then additional research should explore potential causes. A positive correlation would also suggest that complementary treatment options for a laterally leaning uterus should be investigated.

At the 2017 Undergraduate Research Symposium, I presented a low-cost, automated device for influenza detection that was being developed in the Yager lab. After creating, testing, and optimizing this device, we were left with many questions. While certain components of fluid flow in paper are understood, there is much more to learn. In this device alone, three different types of paper were used to run the two-dimensional assay. Current unanswered questions include the following: how do additives affect protein deposition, rehydration, and capture? How much of a dried protein on paper can be rehydrated by a passing solution, and which paper and solution properties affect this value? How do the viscosity and complexity of our patient samples affect the quality of our results? Our overall goal is to determine how to optimize our test line and generate a better understanding for optimization of future diagnostic devices. To make progress towards this goal, my project centers on the development and characterization of a laboratory technique to quantitatively study the effects of these parameters on protein interactions with membrane surfaces and other proteins bound to those membrane surfaces. Improving our understanding of these interactions in membranes will enable better diagnostic device optimization and enable illness detection with lower amounts of infected sample – allowing for earlier detection and better disease treatment. Through the development of this testing apparatus, I have begun to address these questions.

On October 5, 2017, the New York Times revealed sexual harassment allegations against American film producer, Harvey Weinstein. The allegations opened up a floodgate on social media. Actress Alyssa Milano tweeted, “If all the women who have been sexually harassed or assaulted wrote ‘Me too’ as a status, we might give people a sense of the magnitude of the problem.” When networked publics joined in, creating a hashtag, adding their voices, they demonstrated the staggering number of people who have experienced or had something to say about sexual harassment or assault (according to their own definitions). This study examines the cultural and institutional changes created from the hashtag movement. Tweet and retweets with the hashtag #Metoo were collected and analyzed using RiteKit, the social media toolkit. A qualitative analysis was done through tracking the terms “sexual assault,” “sexual misconduct,” “sexual harassment,” or “me too” during the past four months using TheSkimm, an American media company that provides a subscription-only newsletter. The two-part study looked at the use of #MeToo, which created a cultural change as people began to publicly acknowledge sexual harassment and assault as issues and sought to understand how #MeToo lead to substantial and concrete changes, implementations, progress, and/or actions.

In recent years, researchers have dedicated much effort to overcoming the ~250nm spatial resolution limit of light in order to reveal biological details that have been obscured by diffraction. A new form of super-resolution microscopy called expansion microscopy (ExM) has enabled ~65nm image resolution or better by physically expanding fixed specimens in a swellable hydrogel polymer. While ExM allows researchers to achieve high resolution with standard microscopes, the technique requires the use of water-immersion lenses, rather than higher-resolution oil-immersion objective lenses, when imaging more ~5 µm deep due to spherical aberrations caused by refractive index mismatch with oil lenses and water-based hydrogels (n=1.5 vs n=1.3). Oil-immersion lenses also collect more signal than water-immersion lenses, enabling higher signal to noise with oil-immersion lenses under the same excitation conditions. I have developed an index-matching technique that significantly reduces spherical aberrations when imaging with oil lenses and expanded specimens, facilitating improved resolution (~1.12x lateral improvement and 1.5x axial improvement) for depths of up to 25 μm. Expanded specimens are index-matched by simply incubating the specimen in a high-index iohexol solution which equilibrates with the specimen in approximately 18 hours. A modest ~17.5% shrinkage of the hydrogel is produced as a result in the current implementation, which I can in principle compensate for by tuning the hydrogel recipe to achieve a slightly higher expansion factor. I found that the iohexol solution on its own accelerated the rate of fluorophore bleaching and led to some fluorophore quenching. However, the addition of the triplet-state quencher n-propyl-gallate effectively mitigated this chemical bleaching. Ultimately, this iohexol-facilitated index-matching procedure not only combines the utility of ExM and the high collection efficiency of oil immersion objectives but allows researchers to use common chemicals and readily available instruments to obtain images that reveal a previously inaccessible wealth of information.

The North American beaver (Castor canadensis) is a keystone species and ecosystem engineer, capable of modifying the landscape and creating diverse habitats for other species. Through dam-building, beavers impound streamflow and create ponds and wetland complexes behind dams. The presence of beavers has been shown to increase biodiversity, reduce downstream flooding, and improve water quality. While beavers alter landscapes greatly, often in ways beneficial to native habitats and species, their dam-building can also create conflict with human land-use and infrastructure. One way to reduce human-beaver conflict is to adopt non-lethal approaches such as the use of pond leveler devices. Pond levelers, consisting of a pipe through the beaver dam with a cage around the inlet, control the water level of the pond by establishing a desired level while allowing beavers to persist, thereby reducing flooding. Little observational evidence exists on whether pond leveling devices create fish barriers or otherwise impede fish passage for migrating salmon. The objective of this research was to evaluate whether migrating salmon can pass through or around beaver dams fitted with standard pond leveling devices, under high flow conditions. We tested this by observational analysis collected from five pond levelers on Big Spring Creek, King County, Washington during the 2017 autumn salmon migration. Target migrating species include Chinook (Oncorhynchus tshawtscha) and Coho salmon (Oncorhynchus kisutch). During prolonged precipitation events it was determined that stream stage overtopped dams, providing unhindered passage in our study areas. Additionally, during the 2017 migration period, over-dam stream velocity and through-dam velocity (i.e. within the pipe) were passable by salmonids during 100% of the flows we observed.

Alzheimer’s disease (AD) is a progressive degenerative disorder that affects over 5.5 million Americans, resulting in memory loss and cognitive decline over time. AD is characterized by the accumulation of neurofibrillary tangles composed of pathogenic proteins, such as alpha-synuclein. Recent findings suggest that extracellular vesicles (EVs), which primarily function in intercellular communication, may play a critical role in the propagation of these pathogenic proteins in the brain. However, the cellular pathways that load AD-associated toxic proteins into EVs remain unknown. This research project aims to investigate the cellular pathways that influence the biogenesis, secretion, and uptake of EVs carrying AD-associated toxic proteinsusing the powerful genetic model system Saccharomyces cerevisiae. This simple brewer’s yeast has already been proven to be a useful model for understanding AD-related toxicity. When alpha synuclein is overexpressed in S. cerevisiae, it localizes to cell membranes and results in cytoplasmic aggregation that is observed in humans. We purified EVs from yeast expressing human AD-associated toxic proteins (Tau, alpha-synuclein) to establish whether they contain the human transgenes using Western blot analysis. We developed an immunohistochemistry protocol to rapidly quantify the levels of proteins secreted into the extracellular enviroment. We cultured yeast, optimized Western blotting conditions, purified extracellular vesicles, and used imaging techniques in our project.

Computer science and neurobiology have an undeniable entanglement with one another in the 21st century with the advent of advanced artificial intelligence and attempts to understand the black-box model of a brain. However, most neurobiology research has revolved around understanding existing behaviors in large animals, with commonly researched organisms including zebrafish and mice. Introducing novel behaviors to animals in a controlled environment is undeniably attractive in modern neurobiology. With the use of mass data-collection in the online Aquarium lab environment, this study shows the potential to model and produce novel behaviors in Hydra vulgaris (freshwater anemones with simple nervous systems). We aim to create a high-throughput, data-oriented, model for gene-based behavior analysis and editing in neurobiology research. By creating a single controlled environment to perform all research jobs on mass-produced H. vulgaris strains with clockwork husbandry, extraneous factors affecting the organisms are significantly reduced. By taking data on the success of every modification of these Hydra, we can model the effects of specific factors on these animals and progress toward creating a model organism for neurobiology research akin to knockout mice in medicinal genetics. With these methods we have been able to produce a significantly more efficient process of gene editing in Hydra and with several different kinds of DNA integrants. Over the long term, the wide availability of the online Aquarium lab can yield the discovery of unknown trends in genetic knockouts and the discovery of novel behaviors through analysis of all data collected in this controlled environment. Here we highlight successes thus far and future developments necessary to make this system a viable model for gene-based neural engineering over this century.

C. elegans is a prolific model organism that is well established in the field of aging research and age-related diseases. C. elegans can be genetically manipulated to express human toxic proteins associated with neurodegenerative diseases. Its amenability to genetic screening and short lifespan make it an ideal animal model for studying the genetic basis for the neurological health-declines associated with Alzheimer’s disease (AD). Here we introduce new experimental approaches for quantifying the organism-wide impacts of nervous system specific expression of human AD-associated toxic proteins.. The pharynx in C. elegans is an oral pumping structure used in feeding. The pharyngeal nervous system, comprised of only twenty neurons, dictates the rate of pharynx pumping in the animal. Pharyngeal pumping rates have been shown to change and decay with the age of the animal, making it an ideal metric in aging research. The frequency of pharynx pumping can be measured via an electrophysiological recording of the pharynx’s contractions. We plan to use this electropharyngeogram (EPG) as a measure of neurodegeneration to compare wildtype animals with our AD-model mutants. Furthermore, the proper function of extracellular vesicles (ECVs) are thought to be important in the clearance of toxic peptides associated with AD. The only reported phenotype for ECV signaling is the differential development of the cuticle, the animal’s outer-most layer of epidermis. The cuticle’s formation can be assessed through the appearance of the worm’s alae, a set of three lateral stripes running across the animal from head to tail. The appearance of alae has been observed to be different in our AD model mutants, indicating that ECV signaling has been disrupted. Using these AD-model mutants, we aim to establish these novel phenotypes as a means to further investigate the physiological consequences of AD-associated toxic peptide expression.

The field of robotics attempts to replicate human intelligence by harnessing vast amounts of information, and will heavily influence next generation technologies. Of course, in order to operate in an intelligent manner, our robots must be equipped with sensors that can provide informative data. By endowing our robots which such sensors, we can enable them to assist humans that are dependent on others for completing every day tasks, such as opening doors, picking up fallen objects, and pulling wheelchairs. Up until recently, many robots required their sensors to be wired to their main computers, potentially impeding their movement and/or limiting the types of sensors that they can employ. Our work, in particular, focuses on pre-touch sensors, which are sensors mounted to the robot's fingers that allow it to sense an object prior to making contact. This project redeveloped the pre-touch sensing system such that data from up to five sensors is wirelessly transferred to the robot's main computer. There are five micro USB ports connecting to the five fingertip sensors, all of which are controlled by the main USB hub. Vocore, a single-board computer, is incorporated, and this Linux based mini-computer is an essential component for sending the sensor data via Wi-Fi to any selected computer. Additionally, the elimination of wires makes it much easier to reconfigure the form factor of the pre-touch sensing system. As a result, these versatile sensors could be applied to any robot in the world.

The focus of this research is on the abundance of nutrients and which non-toxic heavy metals concentrations in estuarine eelgrass (Zostera marina L.). Eelgrass beds are an important factor in the marine environment, providing a secure habitat for marine life to forge, obtain shelter, spawn, and stabilize the sediment. Also, eelgrass beds reduce costal erosion, meaning healthy eelgrass beds maintain stability in the marine ecosystem. Nutrients and heavy metal concentration is an important element to eelgrass health. Eelgrass plants rely on nutrient abundance to fuel growth, and non-toxic heavy metals in the sediment are absorbed by the plants. Previous research has shown that productive eelgrass beds reduce coastal erosion and cycle carbon dioxide and nutrients into the surrounding ecosystem, acting as a filter for an estuarine habitat. Data collected by the Ocean Research College Academy (ORCA) between 2009 and 2017 were analyzed. It was hypothesized that phosphate and nitrate in the water column would be most abundant during the cold seasons due to high intake of nutrients during the spring and summer, and heavy metal concentration would be more abundant during the spring and summer due to industrial runoff. Results support the original hypothesis, as the data indicate that the highest nutrient abundance occurred December-February each year. Phosphate levels at Site 1 had an abundance of 2.47 [PO₄] μM in December of 2010, which was the highest phosphate level observed. The lowest phosphate abundance throughout the year occurred during the spring of each year. Arsenic, copper, zinc and lead were found to be the most common non-toxic metals concentrated in the eelgrass, which also appeared to have some seasonal influence, likely do to industrial runoff during the spring .

As an airborne virus, influenza A is a widespread threat to global economies and a consistent danger to public health. Through high reassortment and evolutionary rates, influenza is even able to infect those who have been previously vaccinated against the virus. The innate immune system serves as a key first line of defense against this pathogen, with the signaling components, called interferons, driving the production of a potent cellular antiviral response. Studies have indicated that viral populations replete in defective virus particles, virions with a deletion in a portion of their genome, are less efficient at blocking the antiviral response, as shown by increased interferon in the host. Our project seeks to explore this phenomenon of RNA deletions leading to increased interferon expression in host cells by testing the hypothesis that deletions in the three polymerase genes of influenza alone are sufficient to cause an increase in the interferon response. In addition, we are currently testing if mutational deactivation of one of the other genome segments, or absence of such segments, is capable of producing a more robust immune response when combined with polymerase gene deletions. In order to support this analysis, I began by creating pure populations of PA defective influenza particles grown on PA expressing host cells. Similar to results observed by my mentor Dr. Alistair Russell with PB1 and PB2 defective populations, it was found that these PA defective influenza particles were sufficient to induce the interferon response. Recently, I have assisted in the creation of multiple influenza protein expressing cell lines and influenza populations with simultaneous modifications to the HA, NS, and polymerase genes. It is hoped that immune stimulation data derived from these custom viruses, in combination with previous findings, will improve current antiviral therapies and models of the human immune response to influenza.

Obliquity, or axial tilt, is the angle rotational axis and a line perpendicular to the orbital plane. At low obliquity, Earth-like planets will tend to form permanent ice sheets at the poles, if anywhere. However, at high obliquity, the poles receive more sunlight on average than the tropics, and so ice sheets might be expected to form at the equator. We investigate the formation of such “ice-belts” under a range of orbital parameters, obliquities, and host star properties. We find there is a narrow range of semi-major axis and obliquity values that allow for such "ice-belts" to form. First, we experiment with changing the luminosity of the host star to see the effect of changing the stellar flux on ice belt formation, while keeping the relative stellar flux approximate to Earth's, as well as making the appropriate adjustment to the ice-albedo for the host star’s spectrum. Our results suggest that the more luminous the star is, the smaller (and ultimately less stable) the range of ice belt formation becomes. To fully understand the full effect the host star had on ice belt formation, we vary the orbital eccentricity, the spin-axis orientation (precession angle) and the depth of the ocean that can absorb stellar energy (mixing depth). This work suggests that it should not be typical for Earth-like worlds at high obliquity to maintain large ice sheets around main sequence stars.